1. Field of Invention
The present invention relates to droplet structures, emulsions of droplet structures and methods of producing the droplet structures and emulsions; and more particularly to droplet structures, emulsions of droplet structures and methods of producing the droplet structures that are stabilized with block copolymers.
2. Discussion of Related Art
Simple emulsions are dispersions of droplets of one liquid in another immiscible liquid; the droplets are typically formed by applied shear and stabilized against subsequent coalescence by a surfactant that provides an interfacial repulsion (J. Bibette, F. Leal-Calderon, and P. Poulin, Rep. Prog. Phys. 62, 969 (1999)). (All references cited anywhere in any section of this specification are incorporated herein by reference.) Two of the most common types are ‘direct’ oil-in-water (O/W) emulsions and ‘inverse’ water-in-oil (W/O) emulsions. Surfactants are amphiphilic molecules that can take many different forms: ionic (e.g. anionic, cationic, zwitterionic), non-ionic (e.g. ethoxylated alkane chains), and polymeric (e.g. simple, diblock, and triblock polymers). Because they are amphiphilic, surfactants tend to preferentially adsorb onto oil-water interfaces. The relative solubility of the surfactant in the oil and the water, the concentration of the surfactant, and the degree of interfacial repulsion that the surfactant provides once it has adsorbed onto the interfaces are important factors in determining the stability and longevity of emulsions that are formed by an applied shear flow or other sources of non-thermal external stresses that can cause bigger droplet structures to be ruptured into smaller droplet structures.
Beyond simple emulsions, higher levels of topological complexity exist. For instance, a W/O emulsion can be sheared into an aqueous continuous liquid, thereby creating a dispersion of oil droplets which themselves contain smaller water droplets. Through judicious choice of the surfactants, both the ‘inner’ water droplets inside the oil droplets, as well as the larger oil droplets themselves, can remain stable over long periods of time. This type of emulsion is called a water-in-oil-in-water (W/O/W) emulsion. Emulsion systems that have this level of topological complexity are generically called ‘double emulsions’ because, starting from the continuous liquid phase, two oil-water interfacial layers must be penetrated to reach the center of the smallest droplet structure. Indeed, through successive controlled emulsification steps, it is possible to fabricate triple emulsions and even higher topologically ordered multiple emulsions that contain many interfacial layers that must be penetrated in order to reach the center of the smallest droplet structure in the system. A W/O/W double emulsion may have outer oil droplets that each contain only one inner water droplet. However, it is also possible for oil droplets in a W/O/W double emulsion to contain many inner water droplets. Sometimes, this is mistakenly referred to as a “multiple emulsion”. Instead, more properly, it should be referred to as a W/O/W double emulsion that has outer oil droplets that generally each contain a plurality of multiple inner droplets. Two average droplet volume fractions can be used to characterize a double emulsion roughly: the average ‘inner volume fraction’ of water droplets inside the oil droplets, and the average ‘outer volume fraction’ of the W/O droplets that exist in the continuous aqueous solution. Generally, there is a full distribution of radii corresponding to inner water droplets and also a different distribution of radii corresponding to outer water droplets. It can be desirable for these distributions to exhibit monomodal peaks that are fairly sharp, so the droplet sizes are more highly controlled, or ‘uniform’. Another structural aspect that characterizes double emulsions is the probability distribution of the number of inner droplets per outer droplet. Although we focus primarily on creating W/O/W double emulsions (i.e. water-borne double droplets) herein, it is equally possible to create oil-in-water-in-oil (O/W/O) double emulsions that do not have an aqueous continuous phase. For oil-in-water single emulsions and for water-in-oil-in-water double emulsions, φ is typically used to designate the oil volume fraction: the volume of oil contained within the emulsion system divided by the total volume of the emulsion system.
In recent years, two primary pathways, structured microfluidic and sequential emulsification, have provided highly uniform W/O/W double emulsions that typically have average outer droplet diameters greater than about one micron. The first pathway is through relatively low-throughput microfluidic methods. In one implementation of this pathway, a W/O/W emulsion is created using a first cross-channel flow junction to produce water droplets in oil and then using a second cross-channel flow junction to rupture the W/O droplets into a continuous aqueous phase (S. Okushima et al., Langmuir 20, 9905 (2004)). Alternatively, porous glass emulsification and membrane emulsification methods, rather than micromachined fluidic channels, can be used to provide highly uniform W/O emulsions at higher throughput. This implementation permits quite robust incorporation of many inner droplets into double emulsions. A second implementation of microfluidic rupturing is by structuring the flow of an innermost water jet, an intermediate oil jet, and an outermost water jet using microfluidic channels, such that the capillary instability of the inner and outer interfaces occurs simultaneously (A. S. Utada et al., Science 308, 537 (2005)). This method is good for encapsulating objects in the innermost aqueous jet into a W/O/W double emulsion containing a single inner droplet. However, it is significantly more difficult to coordinate the flows so that double emulsions containing a specific number of multiple inner droplets are formed at the desired internal volume fraction. In both of these microfluidic approaches, appropriate surfactants must be present in the liquid phases in order to preserve the stability of the emulsion after formation.
The second pathway is the more traditional form of sequential emulsification without the use of micromachined channels. In sequential emulsification, a W/O emulsion is first created, and then this simple inverse emulsion is, in turn, emulsified into an aqueous surfactant solution using shear (W. Yafei, Z. Tao, and H. Gang, Langmuir 22, 67 (2006)). If desired, both the water and the oil droplets in this W/O/W double emulsion can be size-fractionated to make them monodisperse. Without fractionation, the traditional method can be very high-throughput and can produce many liters per hour. If a high level of monodispersity is desired, then the fractionation necessarily slows down the process. In a variation on this method, a high-throughput approach for making the oil droplets quasi-monodisperse by shearing a premixed double emulsion in a thin gap (C. Goubault et al., Langmuir 17, 5184 (2001)) uses a method previously developed for making monodisperse simple emulsions (T. G. Mason, and J. Bibette, Phys. Rev. Lett. 77, 3481 (1996); T. G. Mason, and J. Bibette, Langmuir 13, 4600 (1997)). As for double emulsions produced using microfluidic methods, the choice of surfactants for sequential emulsification is also important in order to obtain the desired properties of stability and release.
Similar to small molecule surfactants and lipids, synthetic block copolymers are able to self-assemble into ordered nanostructures via microphase separation of the polymeric components (A. J. Link, M. L. Mock, and D. A. Tirrell, Curr Opin Biotech 14, 603 (2003)). However, the ability of block copolymers to assemble into hierarchically structured materials or distinct tertiary structures, similar to those found in biological systems (e.g. proteins), has been limited by the random coiled nature of most common polymers as well as the limited functionality of the polymer domains. Incorporation of elements that encourage hydrogen-bonding (G. A. Silva et al., Science 303, 1352 (2004)), amphiphilicity (D. E. Discher, and A. Eisenberg, Science 297, 967 (2002)), crystallization (G. D. Fasman, Prediction of protein structure and the principles of protein conformation (Plenum Press, New York, 1989), pp. xiii), and liquid crystal formation (D. J. Pochan et al., Macromolecules 35, 5358 (2002)) would all serve to influence structural evolution (J. Rodriguez-Hernandez, and S. Lecommandoux, J Am Chem Soc 127, 2026 (2005)). Increasing the complexity of copolymer sequences (di- to tri- to tetra-blocks, etc.) would also enhance the potential for hierarchical assembly (I. W. Hamley, Soft Matter 1, 36 (2005)). The main limitation in utilizing these strategies is that the synthetic chemistry necessary for preparation of functional, multicomponent block copolymers is a major hindrance due to incompatibilities of different monomers with a given polymerization method (A. J. Link, M. L. Mock, and D. A. Tirrel, Curr Opin Biotech 14, 603 (2003)). Furthermore, since most common synthetic polymers lack the intricate complexity found in biopolymers (e.g. secondary structure, complex functionality and stereochemistry), they may never be able to faithfully mimic the behavior of sell-assemble biological macromolecules. For these reasons, prior to investigating emulsion systems, we have studied the self-assembly of block copolypeptides as synthetic materials that possess the ability to aggregate into specifically defined, functional nanostructures, including vesicles and hydrogels. These non-emulsion materials typically form through interactions between the copolypeptide molecules resulting in “bottom-up” self-assembly. However, the use of synthetic constituents (i.e. non-amino acid monomers) to form synthetic polymer blocks and the use of higher tri-block and multi-block polymer structures are not excluded from some of the general concepts of the current invention.
In work preceding the invention described herein, we focused our efforts on studying the roles of chain length and block composition on the assembly of small, charged diblock copolypeptide amphiphiles, where we utilized the structure directing properties of a rod-like α-helical segment in the hydrophobic domain. Specifically, we prepared and studied the aqueous self-assembly of a series of poly(L-lysine)-b-poly(L-leucine) block copolypeptides, KxLy, where x ranged from 20 to 80, and y ranged from 10 to 30 residues, as well as the poly(L-glutamatic acid)-b-poly(L-leucine) block copolypeptide, E60L20 (E. P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)). The poly(L-lysine.HBr) and poly(L-glutamate-Na+) segments are highly charged polyelectrolytes at neutral pH and dissolve readily in water. In earlier work, we found that samples with high K to L molar ratios (e.g. K180L20) could be dissolved directly into deionized water, yielding transparent hydrogels composed of twisted fibrils (A. P. Nowak et al., Nature 417, 424 (2002)). We reasoned that use of shortened charged segments would relax repulsive polyelectrolyte interactions and allow formation of charged polypeptide membranes. In our first series of copolymers, the size of the oligoleucine domain was held constant at 20 residues, and the oligolysine domain was varied from 20 to 80 residues. Samples were processed by suspending dry polymer in THF/water (1:1) followed by dialysis. Analysis of these assemblies using DIC optical microscopy revealed the presence of large, sheet-like membranes for K20L20, and thin fibrils for K40L20. The K60L20 sample was most promising, as only large vesicular assemblies were observed by differential interference contrast (DIC) microscopy (E. P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)).
The K60L20 polypeptide vesicles obtained directly from dialysis are polydisperse and range in diameter from ca. 5 μm down to 0.8 μm as determined using DIC and DLS (FIG. 1). For applications such as drug delivery via blood circulation, a vesicle diameter of ca. 50 nm to about 100 nm, even up to about 200 nm, is desired. We found that aqueous suspensions of K60L20 vesicles could be extruded through nuclear track-etched polycarbonate (PC) membranes with little loss of polypeptide material. After two passes through a filter, reductions in vesicle diameter to values in close agreement to filter pore size were observed. These results showed that the charged polypeptide vesicles are readily extruded, allowing good control over vesicle diameter in the tens to hundreds of nanometers range (FIG. 1). Dynamic light scattering (DLS) size analysis revealed that the extruded vesicles were also less polydisperse than before extrusion and contained no micellar contaminants. The vesicular morphology was also confirmed through TEM imaging of the sub-micron K60L20 suspensions. The extruded vesicles were monitored for 6 weeks using DLS and were found to be stable since the average diameters did not change for most samples. The vesicles were also found to have high thermal stability. An aqueous suspension of 1 μm vesicles was held at 80° C. for 30 minutes, after which no vesicle disruption could be detected (E. P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)). Only after heating to 100° C. for 30 minutes were the vesicles disrupted, yielding large flat membrane sheets.
Stability of these highly charged polypeptide vesicles in ionic media is important for use in most applications ranging from personal care products to drug delivery. Although the K60L20 vesicles are unstable and cluster at high salt concentrations (>0.5 M), they are stable 100 mM PBS butter as well as serum-free DMEM cell culture media (E. P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)). Addition of serum, which contains anionic proteins, results in vesicle disruption, most likely due to polyion complexation between the serum proteins and the oppositely charged polylysine chains. Accordingly, we found that the negatively charged polypeptide vesicles prepared using E60L20 are stable in DMEM with 10% fetal bovine serum. Based on these results, we believe these charged polypeptide vesicles show potential as encapsulants for water-soluble solutes as an alternative to liposomes. Another feature of these charged polypeptide vesicles is the potential for facile functionalization of the hydrophilic polypeptide chains at the vesicle surface through either chemical conjugation to anine or carboxylate residues, or by careful choice of charged residues. For example, we recently reported the preparation of arginine-leucine (i.e. R60L20) vesicles that are able to readily enter cells due to the many guanidinium groups of the arginine segments (E. P. Holowka et al., Nat Mater 6, 52 (2007)). In this case, the arginine residues played a dual role, where they were both structure directing in vesicle formation, as well as functional for cell binding and entry. The key attributes of block copolypeptides that are advantageous for the design of biomimetic membranes with multifunctional properties are the ability to place structural and functional elements in precise locations within polymer chains. In embodiments of this invention, the copolypeptides populating the interfaces of droplets can also make use of such multifunctional properties, including controlling the morphology and topology of the droplet structures and how they interact with cells and other target materials in applications.
Due to their compartmentalized internal structure, W/O/W double emulsions can provide advantages over simple oil-in-water (O/W) emulsions for encapsulation, such as the ability to carry simultaneously both polar cargoes (such as water-soluble molecules or water dispersable colloids in the inner water droplet) and nonpolar cargoes (such as oil-soluble molecules or oil dispersable colloids in the outer oil droplet), deliver combination therapies of oil-soluble and water-soluble drug molecules to a very specific localized region (e.g. through targeting moieties on molecules that decorate the outer an inner surfaces of the droplets), as well as improved control over temporal release of therapeutic molecules (Pays, K. et al. Double emulsions: how does release occur? Journal of Controlled Release 79, 193-205 (2002); Davis, S. S. & Walker, I. M. Multiple Emulsions as Targetable Delivery Systems. Methods in Enzymology 149, 51-64 (1987); Okochi, H. & Nakano, M. Preparation and evaluation of W/O/W type emulsions containing vancomycin. Advanced Drug Delivery Reviews 45, 5-26 (2000)). The preparation of double emulsions typically requires mixtures of surfactants for stability, and the formation of double nanoemulsions, where both inner and outer droplets are sub-100 nm, has never before been achieved (Garti, N. Double emulsions—Scope, limitations and new achievements. Colloids and Surfaces A—Physicochemical and Engineering Aspects 123, 233-246 (1997); Loscertales, I. G. et al. Micro/nano encapsulation via electrified coaxial liquid jets. Science 295, 1695-1698 (2002); Utada, A. S. et al. Monodisperse double emulsions generated from a microcapillary device. Science 308, 537-541 (2005)).
While offering certain advantages over ordinary O/W emulsions, stable W/O/W emulsions generally do not form spontaneously using a single surfactant and standard emulsification methods according to conventional methods (Garti, N. Double emulsions—Scope, limitations and new achievements. Colloids and Surfaces A—Physicochemical and Engineering Aspects 123, 233-246 (1997); Morais, J. M., Santos, O. D. H., Nunes, J. R. L., Zanatta, C. F., Rocha-Filho, P. A. W/O/W Multiple emulsions obtained by one-step emulsification method and evaluation of the involved variables. Journal of Dispersion Science and Technology 29, 63-69 (2008)). Microfluidics can be used to make double emulsions that are microns in size and highly uniform (Loscertales, I. G. et al. Micro/nano encapsulation via electrified coaxial liquid jets. Science 295, 1695-1698 (2002); Utada, A. S. et al. Monodisperse double emulsions generated from a microcapillary device. Science 308, 537-541 (2005)), yet the throughput can be low compared to commercial processes for making polydisperse single emulsions (Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B. & Graves, S. M. Nanoemulsions: formation, structure, and physical properties. Journal of Physics—Condensed Matter 18, R635-R666 (2006)). Typical methods for making W/O/W emulsions involve a two-step process of first forming an ‘inverse’ water-in-oil (W/O) emulsion, followed by emulsification of this mixture in water using a combination of surfactants (Ficheux, M. F., Bonakdar, L., Leal-Calderon, F. & Bibette, J. Some stability criteria for double emulsions. Langmuir 14, 2702-2706 (1998); Wang, Y. F., Tao, Z. & Gang, H. Structural evolution of polymer-stabilized double emulsions. Langmuir 22, 67-73 (2006); Garti, N. Double emulsions—Scope, limitations and new achievements. Colloids and Surface A—Physicochemical and Engineering Aspects 123, 233-246 (1997); Goubault, C. et al. Shear rupturing of complex fluids: Application to the preparation of quasi-monodisperse water-in-oil-in-water double emulsions. Langmuir 17, 5184-5188 (2001); Okushima, S., Nisisako, T., Torii, T. & Higuchi, T. Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices. Langmuir 20, 9905-9908 (2004)). This process allows control of both inner and outer droplet volumes if the emulsions in both stages are monodisperse, yet this process has not been used to form stable nanoscale droplets (i.e. having both inner and outer droplet diameters that are nanoscale). Moreover, this approach requires a difficult search for surfactant combinations that can co-exist without destabilizing either inner or outer droplet interfaces (Ficheux, M. F., Bonakdar, L., Leal-Calderon, F. & Bibette, J. Some stability criteria for double emulsions. Langmuir 14, 2702-2706 (1998)). Consequently, there is a need for improving stability against evolution of the droplet sizes (e.g. through coalescence and/or coarsening) and reducing droplet sizes in the development of double emulsions for applications (Benichou, A., Aserin, A., Garti, N. Double emulsions stabilized with hybrids of natural polymers for entrapment and slow release of active matters. Advances in Colloid and Interface Science 108-109, 29-41 (2004)).